Agency Response Letter GRAS Notice No. GRN 000345
CFSAN/Office of Food Additive Safety
December 22, 2010
K&L Gates LLP
1601 K Street NW
Washington, DC 20006-1600
Re: GRAS Notice No. GRN 000345
Dear Mr. Yingling:
The Food and Drug Administration (FDA) is responding to the notice, dated June 15, 2010, that you submitted on behalf of DSM Food Specialties (DSM) in accordance with the agency’s proposed regulation, proposed 21 CFR 170.36 (62 FR 18938; April 17, 1997; Substances Generally Recognized as Safe (GRAS); the GRAS proposal). FDA received the notice on June 28, 2010, filed it on June 28, 2010, and designated it as GRAS Notice No. GRN 000345.
The subject of the notice is carboxypeptidase enzyme preparation from modified Aspergillus niger (carboxypeptidase enzyme preparation). The notice informs FDA of the view of DSM that the carboxypeptidase enzyme preparation is GRAS, through scientific procedures, for use as an enzyme in production of cheese to accelerate the ripening process, in production of enzyme-modified cheese as a debittering aid, and in fermented meat to accelerate the development of flavor during the ripening process.
Commercial enzyme preparations that are used in food typically contain an enzyme component, which catalyzes the chemical reaction that is responsible for its technical effect, as well as substances used as stabilizers, preservatives, or diluents. Enzyme preparations may also contain constituents derived from the production organism and manufacturing process. In the notice, DSM provides information about each of these components of the carboxypeptidase enzyme preparation.
The principal activity of carboxypeptidase is the release of C-terminal amino acids from proteins and peptides present in foods. According to the classification system of enzymes established by the International Union of Biochemistry and Molecular Biology, carboxypeptidase is identified by the Enzyme Commission number 3.4.16.x (where x = 1 - 6). Its systematic name is serine-type carboxypeptidase. DSM notes that the A. niger carboxypeptidase is a glycoprotein with a primary sequence of 467 amino acids and a calculated molecular mass of 53 kilodaltons.
DSM states that the production organism, A. niger, is nonpathogenic and nontoxigenic and has a long history of use as a source of enzymes used in food. DSM describes the development of the carboxypeptidase production strain designated as PEG-51A from the host strain designated as ISO-528.
DSM describes the development of the host strain. The A. niger strain NRRL 3122 was initially modified by classical mutagenesis to obtain strain GAM-53. DSM states that strain GAM-53 is fully characterized. To construct the host strain, strain GAM-53 was further modified using molecular biology techniques to create “plug sites” for gene insertion, to inactivate major amylase and protease genes, and to improve enzyme secretion capacity. DSM also states that the host strain has been previously used for construction of the productions strains for asparaginase enzyme preparation (the subject of GRN 000214) and lipase enzyme preparation (the subject of GRN 000296).
To construct the carboxypeptidase production strain, the host strain was transformed with the expression cassette containing the carboxypeptidase gene derived from A. niger strain N400 and a cassette containing the selectable marker gene amdS from Aspergillus nidulans. The amdS gene was subsequently removed to create the marker-free production strain PEG-51A. DSM states that the production strain contains multiple copies of the carboxypeptidase gene, is genetically stable, and contains no antibiotic resistance markers or other heterologous markers. DSM also states that all traces of the production strain are removed during the manufacturing process. DSM notes that specific tests have been performed to confirm that strain PEG-51A does not produce any known toxins under fermentation conditions nor under conditions which are known to induce toxin production in general.
DSM states that carboxypeptidase is produced by cultivation of the production strain using standard fermentation procedures. The fermentation is conducted under controlled conditions and is periodically tested for microbial contamination. Carboxypeptidase is secreted to the fermentation broth and is subsequently recovered by filtration, concentration, and purification, and the purified product is formulated either into a liquid or a spray-dried granulated form. The liquid formulation is stabilized and standardized to a desired carboxypeptidase activity with glycerol. The dried formulation is standardized with maltodextrin. The content of total organic solids (TOS) of the purified enzyme preparation that is used for liquid or dry formulation is approximately 3 percent. The final formulated commercial products contain approximately 1.4 percent TOS. DSM states that the manufacturing process is performed in accordance with current Good Manufacturing Practices. The raw materials used in the fermentation and recovery meet predefined quality standards and the raw materials used for formulation are food grade.
DSM states that that the carboxypeptidase enzyme preparation conforms to the specifications for enzyme preparations described in the 6th edition of the Food Chemicals Codex and to the General Specifications and Considerations for Enzyme Preparations Used in Food Processing established by the FAO/WHO Joint Expert Committee on Food Additives (2006).
DSM states that the carboxypeptidase enzyme preparation will be used at levels of 9.9-100.1 milligrams TOS per kilogram (mg TOS/kg) of the final food. DSM assumes that 100 percent of the enzyme TOS will remain in the final food and estimates a daily intake of the enzyme TOS to be 36 micrograms per kilogram body weight per day (36 µg/kg bw/d). DSM states that carboxypeptidase is expected to be inactivated during the ripening process of cheese and fermented meat and during the pasteurization of enzyme modified cheese.
DSM summarizes the toxicological studies conducted with the purified enzyme concentrate prior to formulation, referred to as the “tox-batch.” The studies include 14-day and 90-day oral toxicity tests in rats, a bacterial reverse mutation assay (Ames test), two in vitro mammalian chromosomal aberration tests, and an in vivo micronucleus test in mice. DSM states that the enzyme concentrate was not toxic in both 14-day and 90-day studies and not mutagenic in the reverse mutation assay. The results of the chromosomal aberration tests show that the enzyme concentrate is not clastogenic to cultured human lymphocytes but may have the potential to disturb mitotic processes and cell cycle progression. To assess this possibility, DSM conducted an in vivo micronucleus test in mice. Based on the results of this test, DSM concluded that the enzyme concentrate is not clastogenic or aneugenic under conditions used in the experiment.
DSM discusses potential allergenicity of enzymes used in food. DSM states that the levels of residual enzymes in food are extremely low and that the potential for sensitization and allergic reactions in consumers is virtually zero. DSM performed an amino acid sequence homology search for carboxypeptidase against known allergens listed in the publicly available Structural Database of Allergenic Proteins (SDAP). The search was performed according to the FAO/WHO guidelines (2001). DSM identified no homology of 35% or greater within a sliding window of 80 amino acids with allergens listed in the SDAP database except for an allergen Api m 9.0101 that occurs in honey bee venom. DSM states that since Api m 9.0101 is not a food allergen this homology is not considered relevant to the assessment of carboxypeptidase. DSM concludes that carboxypeptidase is not a potential food allergen.
Use in Products under USDA Jurisdiction
During its evaluation of GRN 000345, FDA consulted with the Risk and Innovations Management Division, Office of Policy and Program Development, Food Safety and Inspection Service (FSIS) of the United States Department of Agriculture (USDA). Under the Federal Meat Inspection Act, Poultry Products Inspection Act, and the Egg Products Inspection Act, FSIS is responsible for determining the efficacy and suitability of food ingredients in meat, poultry, and egg products as well as prescribing safe conditions of use. Suitability relates to the effectiveness of the ingredient in performing the intended purpose of use and the assurance that the conditions of use will not result in an adulterated product, or one that misleads consumers.
Regarding suitability, FSIS has no objection to the use of carboxypeptidase enzyme preparation at levels of 1.2-6.0 milligrams TOS/kg of fermented meat. The enzyme, however, will need to be listed as Carboxypeptidase (CPG) enzyme or “enzyme” in the ingredients statement on the label of the products in which it is used as an ingredient. Further questions regarding regulatory guidance from FSIS about the use of carboxypeptidase enzyme preparation in fermented meat products should be directed to Ms. Valeria Jefferson, Risk and Innovations Management Division, Office of Policy and Program Development, Food Safety and Inspection Service, 5601 Sunnyside Avenue, Mail Stop 5271, Beltsville, MD 20705-5271.
Section 301(ll) of the Federal Food, Drug, and Cosmetic Act (FFDCA)
Section 301(ll) of the FFDCA prohibits the introduction or delivery for introduction into interstate commerce of any food that contains a drug approved under section 505 of the FFDCA, a biological product licensed under section 351 of the Public Health Service Act, or a drug or a biological product for which substantial clinical investigations have been instituted and their existence made public, unless one of the exemptions in section 301(ll)(1)-(4) applies. In its review of DSM’s notice that the carboxypeptidase enzyme preparation is GRAS for the intended uses, FDA did not consider whether section 301(ll) or any of its exemptions apply to foods containing the carboxypeptidase enzyme preparation. Accordingly, this response should not be construed to be a statement that foods that contain the carboxypeptidase enzyme preparation, if introduced or delivered for introduction into interstate commerce, would not violate section 301(ll).
Based on the information provided by DSM, as well as other information available to FDA, the agency has no questions at this time regarding DSM’s conclusion that the carboxypeptidase enzyme preparation is GRAS under the intended conditions of use. The agency has not, however, made its own determination regarding the GRAS status of the subject use of the carboxypeptidase enzyme preparation. As always, it is the continuing responsibility of DSM to ensure that food ingredients that the firm markets are safe, and are otherwise in compliance with all applicable legal and regulatory requirements.
In accordance with proposed 21 CFR 170.36(f), a copy of the text of this letter responding to GRN 000345, as well as a copy of the information in this notice that conforms to the information in the GRAS exemption claim (proposed 21 CFR 170.36(c)(1)), is available for public review and copying at www.fda.gov/grasnoticeinventory.
Mitchell A. Cheeseman, Ph.D.
Office of Food Additive Safety
Center for Food Safety and Applied Nutrition
cc: Ms. Valeria Jefferson
Risk and Innovations Management Division
Office of Policy and Program Development
Food Safety and Inspection Service
George Washington Carver Center (GWCC)
5601 Sunnyside Avenue, Mail STOP 5271
Beltsville, MD 20705-5271